| 1. |
- Fischer, Benjamin, et al.
(författare)
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A complete workflow for the differentiation and the dissociation of hiPSC-derived cardiospheres
- 2018
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Ingår i: Stem Cell Research. - : Elsevier. - 1873-5061 .- 1876-7753. ; 32, s. 65-72
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Tidskriftsartikel (refereegranskat)abstract
- Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) are an invaluable tool for both basic and translational cardiovascular research. The potential that these cells hold for therapy, disease modeling and drug discovery is hampered by several bottlenecks that currently limit both the yield and the efficiency of cardiac induction. Here, we present a complete workflow for the production of ready-to-use hiPSC-CMs in a dynamic suspension bioreactor. This includes the efficient and highly reproducible differentiation of hiPSCs into cardiospheres, which display enhanced physiological maturation compared to static 3D induction in hanging drops, and a novel papain-based dissociation method that offers higher yield and viability than the broadly used dissociation reagents TrypLE and Accutase. Molecular and functional analyses of the cardiomyocytes reseeded after dissociation confirmed both the identity and the functionality of the cells, which can be used in down-stream applications, either as monolayers or spheroids.
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| 2. |
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| 3. |
- Lindberg, Frida W., et al.
(författare)
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Controlled Surface Silanization for Actin-Myosin and Biocompatibility of New Polymer Resists
- 2018
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Ingår i: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 34:30, s. 8777-8784
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Tidskriftsartikel (refereegranskat)abstract
- Molecular motor-based nanodevices require organized cytoskeletal filament guiding along motility-promoting tracks, confined by motility-inhibiting walls. One way to enhance motility quality on the tracks, particularly in terms of filament velocity but also the fraction of motile filaments, is to optimize the surface hydrophobicity. We have investigated the potential to achieve this for the actin myosin II motor system on trimethylchlorosilane (TMCS)-derivatized SiO2 surfaces to be used as channel floors in nanodevices. We have also investigated the ability to supress motility on two new polymer resists, TU7 (for nanoimprint lithography) and CSAR 62 (for electron beam and deep UV lithography), to be used as channel walls. We developed a chemical-vapor deposition tool for silanizing SiO2 surfaces in a controlled environment to achieve different surface hydrophobicities (measured by water contact angle). In contrast to previous work, we were able to fabricate a wide range of contact angles by varying the silanization time and chamber pressure using only one type of silane. This resulted in a significant improvement of the silanization procedure, producing a predictable contact angle on the surface and thereby predictable quality of the heavy meromyosin (HMM)-driven actin motility with regard to velocity. We observed a high degree of correlation between the filament sliding velocity and contact angle in the range 10-86 degrees, expanding the previously studied range. We found that the sliding velocity on TU7 surfaces was superior to that on CSAR 62 surfaces despite similar contact angles. In addition, we were able to suppress the motility on both TU7 and CSAR 62 by plasma oxygen treatment before silanization. These results are discussed in relation to previously proposed surface adsorption mechanisms of HMM and their relationship to the water contact angle. Additionally, the results are considered for the development of actin-myosin based nanodevices with superior performance with respect to actin-myosin functionality.
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| 4. |
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| 5. |
- Meinecke, Christoph R., et al.
(författare)
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Nanolithographic Fabrication Technologies for Network-Based Biocomputation Devices
- 2023
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Ingår i: Materials. - : MDPI. - 1996-1944. ; 16:3
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Tidskriftsartikel (refereegranskat)abstract
- Network-based biocomputation (NBC) relies on accurate guiding of biological agents through nanofabricated channels produced by lithographic patterning techniques. Here, we report on the large-scale, wafer-level fabrication of optimized microfluidic channel networks (NBC networks) using electron-beam lithography as the central method. To confirm the functionality of these NBC networks, we solve an instance of a classical non-deterministic-polynomial-time complete ("NP-complete") problem, the subset-sum problem. The propagation of cytoskeletal filaments, e.g., molecular motor-propelled microtubules or actin filaments, relies on a combination of physical and chemical guiding along the channels of an NBC network. Therefore, the nanofabricated channels have to fulfill specific requirements with respect to the biochemical treatment as well as the geometrical confienement, with walls surrounding the floors where functional molecular motors attach. We show how the material stack used for the NBC network can be optimized so that the motor-proteins attach themselves in functional form only to the floor of the channels. Further optimizations in the nanolithographic fabrication processes greatly improve the smoothness of the channel walls and floors, while optimizations in motor-protein expression and purification improve the activity of the motor proteins, and therefore, the motility of the filaments. Together, these optimizations provide us with the opportunity to increase the reliability of our NBC devices. In the future, we expect that these nanolithographic fabrication technologies will enable production of large-scale NBC networks intended to solve substantially larger combinatorial problems that are currently outside the capabilities of conventional software-based solvers.
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| 6. |
- Rahman, Mohammad A., et al.
(författare)
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Comparative analysis of widely used methods to remove nonfunctional myosin heads for the in vitro motility assay
- 2018
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Ingår i: Journal of Muscle Research and Cell Motility. - : Springer. - 0142-4319 .- 1573-2657. ; 39:5-6, s. 175-187
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Tidskriftsartikel (refereegranskat)abstract
- The in vitro motility assay allows studies of muscle contraction through observation of actin filament propulsion by surface-adsorbed myosin motors or motor fragments isolated from muscle. A possible problem is that motility may be compromised by nonfunctional, "dead", motors, obtained in the isolation process. Here we investigate the effects on motile function of two approaches designed to eliminate the effects of these dead motors. We first tested the removal of heavy meromyosin (HMM) molecules with ATP-insensitive "dead" heads by pelleting them with actin filaments, using ultracentrifugation in the presence of 1 mM MgATP ("affinity purification"). Alternatively we incubated motility assay flow cells, after HMM surface adsorption, with non-fluorescent "blocking actin" (1 µM) to block the dead heads. Both affinity purification and use of blocking actin increased the fraction of motile filaments compared to control conditions. However, affinity purification significantly reduced the actin sliding speed in five out of seven experiments on silanized surfaces and in one out of four experiments on nitrocellulose surfaces. Similar effects on velocity were not observed with the use of blocking actin. However, a reduced speed was also seen (without affinity purification) if HMM or myosin subfragment 1 was mixed with 1 mM MgATP before and during surface adsorption. We conclude that affinity purification can produce unexpected effects that may complicate the interpretation of in vitro motility assays and other experiments with surface adsorbed HMM, e.g. single molecule mechanics experiments. The presence of MgATP during incubation with myosin motor fragments is critical for the complicating effects.
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| 7. |
- Rahman, Mohammad A., et al.
(författare)
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Regeneration of Assembled, Molecular-Motor-Based Bionanodevices
- 2019
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Ingår i: Nano Letters. - : American Chemical Society (ACS). - 1530-6984 .- 1530-6992. ; 19:10, s. 7155-7163
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Tidskriftsartikel (refereegranskat)abstract
- The guided gliding of cytoskeletal filaments, driven by biomolecular motors on nano/microstructured chips, enables novel applications in biosensing and biocomputation. However, expensive and time-consuming chip production hampers the developments. It is therefore important to establish protocols to regenerate the chips, preferably without the need to dismantle the assembled microfluidic devices which contain the structured chips. We here describe a novel method toward this end. Specifically, we use the small, nonselective proteolytic enzyme, proteinase K to cleave all surface-adsorbed proteins, including myosin and kinesin motors. Subsequently, we apply a detergent (5% SDS or 0.05% Triton X100) to remove the protein remnants. After this procedure, fresh motor proteins and filaments can be added for new experiments. Both, silanized glass surfaces for actin-myosin motility and pure glass surfaces for microtubule-kinesin motility were repeatedly regenerated using this approach. Moreover, we demonstrate the applicability of the method for the regeneration of nano/microstructured silicon-based chips with selectively functionalized areas for supporting or suppressing gliding motility for both motor systems. The results substantiate the versatility and a promising broad use of the method for regenerating a wide range of protein-based nano/microdevices.
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| 8. |
- Reuther, Cordula, et al.
(författare)
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Comparison of actin- and microtubule-based motility systems for application in functional nanodevices
- 2021
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Ingår i: New Journal of Physics. - : IOP Publishing. - 1367-2630. ; 23:7
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Tidskriftsartikel (refereegranskat)abstract
- Over the last 25 years, extensive progress has been made in developing a range of nanotechnological applications where cytoskeletal filaments and molecular motors are key elements. This includes novel, highly miniaturized lab on a chip systems for biosensing, nanoseparation etc but also new materials and parallel computation devices for solving otherwise intractable mathematical problems. For such approaches, both actin-based and microtubule-based cytoskeletal systems have been used. However, in accordance with their different cellular functions, actin filaments and microtubules have different properties and interaction kinetics with molecular motors. Therefore, the two systems obviously exhibit different advantages and encounter different challenges when exploited for applications. Specifically, the achievable filament velocities, the capability to guide filaments along nanopatterned tracks and the capability to attach and transport cargo differ between actin- and microtubule-based systems. Our aim here is to systematically elucidate these differences to facilitate design of new devices and optimize future developments. We first review the cellular functions and the fundamental physical and biochemical properties of actin filaments and microtubules. In this context we also consider their interaction with molecular motors and other regulatory proteins that are of relevance for applications. We then relate these properties to the advantages and challenges associated with the use of each of the motor-filament systems for different tasks. Finally, fundamental properties are considered in relation to some of the most interesting future development paths e.g. in biosensing and biocomputation.
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| 9. |
- Reuther, Cordula, et al.
(författare)
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Molecular motor-driven filament transport across three-dimensional, polymeric micro-junctions
- 2021
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Ingår i: New Journal of Physics. - : Institute of Physics Publishing (IOPP). - 1367-2630. ; 23:12
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Tidskriftsartikel (refereegranskat)abstract
- Molecular motor-driven filament systems have been extensively explored for biomedical and nanotechnological applications such as lab-on-chip molecular detection or network-based biocomputation. In these applications, filament transport conventionally occurs in two dimensions (2D), often guided along open, topographically and/or chemically structured channels which are coated by molecular motors. However, at crossing points of different channels the filament direction is less well determined and, though crucial to many applications, reliable guiding across the junction can often not be guaranteed. We here present a three-dimensional (3D) approach that eliminates the possibility for filaments to take wrong turns at junctions by spatially separating the channels crossing each other. Specifically, 3D junctions with tunnels and overpasses were manufactured on glass substrates by two-photon polymerization, a 3D fabrication technology where a tightly focused, femtosecond-pulsed laser is scanned in a layer-to-layer fashion across a photo-polymerizable inorganic-organic hybrid polymer (ORMOCER(R)) with mu m resolution. Solidification of the polymer was confined to the focal volume, enabling the manufacturing of arbitrary 3D microstructures according to computer-aided design data. Successful realization of the 3D junction design was verified by optical and electron microscopy. Most importantly, we demonstrated the reliable transport of filaments, namely microtubules propelled by kinesin-1 motors, across these 3D junctions without junction errors. Our results open up new possibilities for 3D functional elements in biomolecular transport systems, in particular their implementation in biocomputational networks.
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| 10. |
- Salhotra, Aseem
(författare)
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Actomyosin in biocomputation
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- There exist complex mathematical problems that are important in real world applications such as weather prediction, molecular modelling, network route optimization and more. In general, such problems are solved using supercomputers with higher computing efficiency but this also consumes high energy along with high production and maintenance cost. Network-based biocomputation (NBC) is an alternate computing approach, now at development stage, that can perform parallel computing in a highly energy efficient manner. Actin and myosin constitute one type of molecular motor system that has been utilized for the development of NBC. These proteins are key components in the sarcomere, the smallest functional unit of muscle and their interactions that underlie muscle contraction are powered by the cellular fuel adenosine triphosphate (ATP). To solve larger complex problems using actin-myosin based NBC, factors such as maintained biological function and longevity of operation are essential for practical relevance. In this thesis, the in vitro motility assay (IVMA) has been used as a central method to study actomyosin function and its operation within NBC devices. In the IVMA, actin filaments are propelled by myosin motors that are immobilized on functionalized surfaces in a flow cell. With the aim to improve motile fraction by reducing the interaction between actin and non-functional motor heads in the IVMA, two known methods were quantitatively compared in paper I, the affinity purification and the blocking actin method. Both approaches significantly improved the motile fraction to above 90% but affinity purification, due to the presence of ATP during incubation, induced significant reduction in sliding velocity, not seen with blocking actin. In paper III, critical parameters in the actomyosin IVMA system were investigated allowing us to extensively improve function and longevity, including: biocompatibility of flow cell components, effects of air exposure with oxygen scavenging and nanofabrication parameters such as plasma etching type and time, process of resist development, and surface silanization time. The above developments together with optimized network encoding of the problems enabled us (paper IV) to solve four instances of 3-SAT problem encoded in NBC with 99% probability of satisfiability. In parallel, (paper II) a method have been developed to recycle the surfaces with immobilized motor proteins by treatment of proteinase-K enzyme and detergent. This will allow re-cycling of advanced NBC chips. Finally, with aim to develop programmable gating for NBC, attempts have been made towards the integration of engineered light sensitive myosin XI motors with nanofabricated devices made up of Au/SiO2, SiO2/polymer and glass/polymer (paper V). In addition important factors such as standardized motor density, limiting of air exposure and longevity function have been optimized in the use of light sensitive motors.Overall, this thesis reports critical insights for the upscaling of actomyosin based NBC. Described results, are also useful for the development of actomyosin based nanotechnological applications such as biosensing or diagnostics and other fundamental studies based on single molecule or drug testing.
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| 11. |
- Salhotra, Aseem, et al.
(författare)
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Exploitation of Engineered Light-Switchable Myosin XI for Nanotechnological Applications
- 2023
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Ingår i: ACS Nano. - : American Chemical Society (ACS). - 1936-0851 .- 1936-086X. ; 17:17, s. 17233-17244
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Tidskriftsartikel (refereegranskat)abstract
- For certain nanotechnological applications of the contractile proteins actin and myosin, e.g., in biosensing and network-based biocomputation, it would be desirable to temporarily switch on/off motile function in parts of nanostructured devices, e.g., for sorting or programming. Myosin XI motor constructs, engineered with a light-switchable domain for switching actin motility between high and low velocities (light-sensitive motors (LSMs) below), are promising in this regard. However, they were not designed for use in nanotechnology, where longevity of operation, long shelf life, and selectivity of function in specific regions of a nanofabricated network are important. Here, we tested if these criteria can be fulfilled using existing LSM constructs or if additional developments will be required. We demonstrated extended shelf life as well as longevity of the actin-propelling function compared to those in previous studies. We also evaluated several approaches for selective immobilization with a maintained actin propelling function in dedicated nanochannels only. Whereas selectivity was feasible using certain nanopatterning combinations, the reproducibility was not satisfactory. In summary, the study demonstrates the feasibility of using engineered light-controlled myosin XI motors for myosin-driven actin transport in nanotechnological applications. Before use for, e.g., sorting or programming, additional work is however needed to achieve reproducibility of the nanofabrication and, further, optimize the motor properties.
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| 12. |
- Salhotra, Aseem, et al.
(författare)
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Prolonged function and optimization of actomyosin motility for upscaled network-based biocomputation
- 2021
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Ingår i: New Journal of Physics. - : IOP Publishing. - 1367-2630. ; 23:8
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Tidskriftsartikel (refereegranskat)abstract
- Significant advancements have been made towards exploitation of naturally available molecular motors and their associated cytoskeletal filaments in nanotechnological applications. For instance, myosin motors and actin filaments from muscle have been used with the aims to establish new approaches in biosensing and network-based biocomputation. The basis for these developments is a version of the in vitro motility assay (IVMA) where surface-adsorbed myosin motors propel the actin filaments along suitably derivatized nano-scale channels on nanostructured chips. These chips are generally assembled into custom-made microfluidic flow cells. For effective applications, particularly in biocomputation, it is important to appreciably prolong function of the biological system. Here, we systematically investigated potentially critical factors necessary to achieve this, such as biocompatibility of different components of the flow cell, the degree of air exposure, assay solution composition and nanofabrication methods. After optimizing these factors we prolonged the function of actin and myosin in nanodevices for biocomputation from 60 min. In addition, we demonstrated that further optimizations could increase motility run times to >20 h. Of great importance for the latter development was a switch of glucose oxidase in the chemical oxygen scavenger system (glucose oxidase-glucose-catalase) to pyranose oxidase, combined with the use of blocking actin (non-fluorescent filaments that block dead motors). To allow effective testing of these approaches we adapted commercially available microfluidic channel slides, for the first time demonstrating their usefulness in the IVMA. As part of our study, we also demonstrate that myosin motor fragments can be stored at -80 degrees C for more than 10 years before use for nanotechnological purposes. This extended shelf-life is important for the sustainability of network-based biocomputation.
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| 13. |
- Surendiran, Pradheebha, et al.
(författare)
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Solving Exact Cover Instances with Molecular-Motor-Powered Network-Based Biocomputation
- 2022
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Ingår i: ACS Nanoscience Au. - : American Chemical Society (ACS). - 2694-2496 .- 2694-2496.
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Tidskriftsartikel (refereegranskat)abstract
- Information processing by traditional, serial electronic processors consumes an ever-increasing part of the global electricity supply. An alternative, highly energy efficient, parallel computing paradigm is network-based biocomputation (NBC). In NBC a given combinatorial problem is encoded into a nanofabricated, modular network. Parallel exploration of the network by a very large number of independent molecular-motor-propelled protein filaments solves the encoded problem. Here we demonstrate a significant scale-up of this technology by solving four instances of Exact Cover, a nondeterministic polynomial time (NP) complete problem with applications in resource scheduling. The difficulty of the largest instances solved here is 128 times greater in comparison to the current state of the art for NBC.
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| 14. |
- Ušaj, Marko, et al.
(författare)
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Single molecule turnover of fluorescent ATP by myosin and actomyosin unveil elusive enzymatic mechanisms
- 2021
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Ingår i: Communications Biology. - : Nature Publishing Group. - 2399-3642. ; 4:1, s. 1-12
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Tidskriftsartikel (refereegranskat)abstract
- Benefits of single molecule studies of biomolecules include the need for minimal amounts of material and the potential to reveal phenomena hidden in ensembles. However, results from recent single molecule studies of fluorescent ATP turnover by myosin are difficult to reconcile with ensemble studies. We found that key reasons are complexities due to dye photophysics and fluorescent contaminants. After eliminating these, through surface cleaning and use of triple state quenchers and redox agents, the distributions of ATP binding dwell times on myosin are best described by 2 to 3 exponential processes, with and without actin, and with and without the inhibitor para-aminoblebbistatin. Two processes are attributable to ATP turnover by myosin and actomyosin respectively, whereas the remaining process (rate constant 0.2-0.5 s(-1)) is consistent with non-specific ATP binding to myosin, possibly accelerating ATP transport to the active site. Finally, our study of actin-activated myosin ATP turnover without sliding between actin and myosin reveals heterogeneity in the ATP turnover kinetics consistent with models of isometric contraction.
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| 15. |
- Zhu, Jingyuan, et al.
(författare)
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Solving the 3-Satisfiability Problem Using Network-Based Biocomputation
- 2022
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Ingår i: Advanced Intelligent Systems. - : John Wiley & Sons. - 2640-4567. ; 4:12
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Tidskriftsartikel (refereegranskat)abstract
- The 3-satisfiability Problem (3-SAT) is a demanding combinatorial problem that is of central importance among the nondeterministic polynomial (NP) complete problems, with applications in circuit design, artificial intelligence, and logistics. Even with optimized algorithms, the solution space that needs to be explored grows exponentially with the increasing size of 3-SAT instances. Thus, large 3-SAT instances require excessive amounts of energy to solve with serial electronic computers. Network-based biocomputation (NBC) is a parallel computation approach with drastically reduced energy consumption. NBC uses biomolecular motors to propel cytoskeletal filaments through nanofabricated networks that encode mathematical problems. By stochastically exploring possible paths through the networks, the cytoskeletal filaments find possible solutions. However, to date, no NBC algorithm for 3-SAT has been available. Herein, an algorithm that converts 3-SAT into an NBC-compatible network format is reported and four small 3-SAT instances (with up to three variables and five clauses) using the actin-myosin biomolecular motor system are experimentally solved. Because practical polynomial conversions to 3-SAT exist for many important NP complete problems, the result opens the door to enable NBC to solve small instances of a wide range of problems.
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